This invention relates to a promoting agent for bone formation which comprises a peptide or polypeptide containing the amino acid sequence consisting of ArgGlyAsp (hereinafter referred to as RGD sequence) in the molecule. Further, the invention relates to a prophylactic and therapeutic method for bone fracture utilizing a promoting agent for bone formation comprising the peptide or polypeptide containing the said RGD sequence. Also, the invention relates to a novel cyclic peptide containing the said RGD sequence. And further, it relates to a promoting agent for bone formation which comprises a compound represented by the general formulae (IX), (X), (XI) and (XII).
Bone is composed of outer cortical bone and inner trabecular bone. The function of bone in living body is to maintain a given shape as a skeleton and to store various inorganic substances such as calcium, phosphoric acid and the like. Bone may apparently appear to be a tissue with less variability, but actually old bone is adsorbed and instead new bone is formed. This is usually referred to as bone reformation. The bone reformation can be accomplished by coupling of osteoclasts controlling bone adsorption with osteoblasts controlling bone formation as both can primarily participate in therein. It has been recently elucidated that the function of osteoblasts is not limited to bone formation solely, but it is related to differentiation and activation of osteoclasts and osteoblasts may play a role as a control center in cellular bone reconstruction.
Those diseases generally referred to as bone metabolism diseases may include osteoporosis, Behcet disease, osteomalacia, hyperostosis and osteopetrosis. Among them, osteoporosis is the most frequently developed disease and frequency of its occurrence appears to increase with senescence so that the diagnosis and effective therapy thereof have been earnestly desired.
The bone metabolism diseases mean those diseases wherein bone cells have specific metabolic abnormalities in any bone tissues. The present inventors have made earnest studies to find out a promoting factor for bone formation using a cultural assay and finally completed this invention.
Integrin may participate in the interaction between cells and cells or between cells and extracellular matrices and play an important role in wound healing, development, immunization, hemostasis or metastasis. Integrin superfamily is an xcex1,xcex2-heterodimer group found on the cell surface and may combine extracellular ligands and cytoskeleton. All integrins are heterodimers and each sub-unit is extracellular by 90% and may have a long membrane permeable domain and a short intracellular domain. The extracellular domain is bound with the extracellular matrices or ligands of the cell surface, while the intracellular domain is bound with cytoskeleton proteins. Bone matrices such as osteopontin, bone sialoglycoproteins, thrombospondin, fibronectin and vitronectin are found in bone and all proteins have been found to have the RGD sequence. Recently, osteoclasts have been found to have integrin xcex1Vxcex23 and xcex12xcex21 in the cell membrane surface (Davies J. et al., J. Cell Biol., Vol.1, 109, p.1817, 1989 and Zambonin Z. A. et al., Connect. Tissue Res., Vol.20, p.143, 1989). From the facts that bone absorption by osteoclasts can be inhibited by the action of an antibody to integrin (Davies J. et al., J. Cell Biol., Vol.109, p.1817, 1989), that bone absorption by rat osteoclasts can be inhibited by synthetic GRGDSP peptide (GlyArgGlyAspSerPro)(Horton M. A. et al., Exp. Cell Res., Vol.195, p.368, 1991), and further that echstatin, a protein derived from snake venom and having the RGD sequence and a platelet aggregation inhibiting activity, a synthetic GdRGDSP peptide and a cyclic synthetic GPenGRGDSPCA peptide can inhibit bone absorption by mouse osteoclasts and GdRGDSP peptide can inhibit the formation of tartaric acid-resistant and phosphatase-positive multinuclear osteoclasts (Gabri V. D. P. et al., J. Bone Miner. Res., Vol.9, p.1021, 1994), it is suggested that recognition and adhesion of bone matrices by integrin and related cytoskeleton does deeply participate in the development of bone absorption function of osteoclasts.
It may be then considered that the adhesion of cell matrices between osteoblasts and bone matrices would be caused by the adhesion mechanism via collagen and fibronectin in the bone matrices and xcex21 integrin of osteoblasts. Also, it would be possible in the adhesion mechanism between heterocytes that the cell adhesion of osteoblasts with osteoclasts can be performed via fibronectin as both xcex23 integrin of osteoclasts and xcex21 integrin in osteoblasts may be a receptor for fibronectin. However, it has not yet been suggested that the disintegrin family including echstatin or kistrin (William R. G. et al., Protein Science, Vol.2, p.1749, 1993) and the RGD peptide show a promoting action for bone formation.
It is the object of this invention to provide a promoting agent for bone formation, a therapeutic method for bone formation and a process for preparing a promoting agent for bone formation.
Bone formation can be promoted by administering a peptide or polypeptide containing the RGD sequence in the molecule or a biologically acceptable salt thereof to patients. The peptides or polypeptide containing the RGD sequence in the molecule may illustratively include kistrin, echstatin, a peptide represented by Gly-Arg-Gly-Asp-Ser (hereinafter referred to as GRGDS) (SEQ ID NO: 1), a compound represented by the general formula (I) 
wherein R1, R2, R3, R4, R5 and R6 may be the same or different and each represents one selected from the group consisting of a hydrogen atom; an alkyl group of 1-8 carbon atoms optionally substituted with one selected from the group consisting of a hydroxy group, a carboxy group, a cycloalkyl group of 3-10 carbon atoms optionally substituted with a hydroxy group, and an aryl group of 6-12 carbon atoms optionally substituted with a hydroxy group ; a cycloalkyl group of 3-10 carbon atoms optionally substituted with hydroxy group and an aryl group of 6-12 carbon atoms optionally substituted with a hydroxy group, R7 and R8 may be the same of different and each represents a group selected from the group consisting of a hydroxy group, an alkoxy group of 1-8 carbon atoms, an alkenyloxy group of 2-12 carbon atoms, a cycloalkyloxy group of 3-10 carbon atoms and an aryloxy group of 6-12 carbon atoms, and X represents S or SO2 and a compound represented by the general formula (II) 
wherein R9, R10, R11 and R12 may be the same or different and each represents one selected from the group consisting of a hydrogen atom, an alkyl group of 1-8 carbon atoms, a cycloalkyl group of 3-10 carbon atoms and an aryl group of 6-12 carbon atoms optionally substituted with a hydroxy group, R13, R14 and R15 may be the same or different and each represents a group selected from the group consisting of a hydroxy group, an alkoxy group of 1-8 carbon atoms, an alkenyloxy group of 2-12 carbon atoms, a cycloalkyloxy group of 3-10 carbon atoms and an aryloxy group of 6-12 carbon atoms, and X represents S or SO.
And, this invention provides a new compound represented by the formula (II).
Also , this invention is directed to a therapeutic method for promoting bone formation which comprises administering to patients a compound represented by the general formula (IX) 
wherein R16 represents xe2x80x94N(R20)2, xe2x80x94C(xe2x95x90NH)xe2x80x94NH2, xe2x80x94NHxe2x80x94C(xe2x95x90NH)xe2x80x94NH2 or xe2x80x94COxe2x80x94NHxe2x80x94C(xe2x95x90NH)xe2x80x94NH2 (in which R20 independently represents a hydrogen atom or an alkyl group of 1-4 carbon atoms optionally substituted with a phenyl group), R17 represents a hydrogen atom or an alkyl group of 1-4 carbon atoms optionally substituted with a phenyl group, R18 represents a hydrogen atom, an alkyl group of 1-4 carbon atoms, a phenyl group optionally substituted with a methoxy group or xe2x80x94COR21 (in which R21 represents xe2x80x94OH, xe2x80x94NH2, xe2x80x94NHxe2x80x94(CH2)2-phenyl, an alkoxy group of 1-3 carbon atoms, a benzyloxy group, Pro or Aoc), R19 represents an alkyl group of 1-5 carbon atoms optionally substituted with a substituent selected from the group consisting of OH, xe2x80x94NH2, xe2x80x94CONH2, cyclohexyl, phenyl, naphthyl, indolyl or adamantyl, a methyl group substituted with xe2x80x94COOH and xe2x80x94NHCOOCH2-phenyl group, a cyclohexyl group optionally substituted with a methoxy group or an aryl group of 6-10 carbon atoms optionally substituted with a methoxy group, provided that R18 and R19 together with the carbon atoms to which they are attached may form adamantyl, naphthyl or fluorenyl, Y represents xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 or a direct bond, a represents 1, 2 or 3, b represents 1 or 2, c represents 0 or 1 and d represents 0 or 1, a compound represented by the general formula(X) 
wherein R22 represents xe2x80x94N(R23)2, xe2x80x94C(xe2x95x90NH)NH2, xe2x80x94NHxe2x80x94C(xe2x95x90NH)xe2x80x94NH2 or xe2x80x94COxe2x80x94NHxe2x80x94C(xe2x95x90NH)xe2x80x94NH2 (in which R23 independently represents a hydrogen atom or an alkyl group of 1-4 carbon atoms optionally substituted with a phenyl group) and e represents 2-6, a compound of the formula (XI) 
or
a compound of the formula (XII) 
or a biologically acceptable salt thereof.
In the above formulae, Pro and Aoc represent respecively 
Kistrin and echstatin are proteins having molecular eights of about 7,300 and about 5,400, respectively, and they are disclosed, for instance, Protein Science (1993), 2, 1749-1755. The GRGDS (SEQ ID NO: 1) may be easily synthesized according to a conventional peptide synthesis. The compounds of the general formula (I) are disclosed in U.S. Pat. No. 5,384,309 and their representative compounds may be illustrated by the formulae (III)-(VI). 
The compounds of the general formula (II) are novel and may be prepared according to the method as described in Barker et al., J. Med. Chem., 1992, 35,2040. Their representative compounds may be illustrated by the formulae (VII) and (VIII). 
Illustrative compounds of the general formula (IX) are as recited below. 
Illustrative compound of the general formula (XI) is as recited below.
Compound No. 64
Illustrative compound of the general formula (XII) is as recited below.
Compound No. 65
The compounds of the general formulae (IX), (X), (XI) and (XII) are known and may be synthesized according to the methods disclosed in EP-A0499079, EP-A0530505, EP-A0566919, WO 95/14008, EP-A0528586 and WO 93/19046. When kistrin or echstatin is to be administered to human as a promoting agent for bone formation, a daily dose of 0.001-100 xcexcg/kg body weight, preferably 0.01-10 xcexcg/kg body weight, is used. In the case of the GRGDS (SEQ ID NO: 1), a daily dose of 0.001-10 mg/kg body weight, preferably 0.01-1 mg/kg body weight, is used. In the case of the compound represented by the general formula (I), (II), (IX), (X), (XI) or (XII), a daily dose of 0.001-10 mg/kg body weight, preferably 0.01-1 mg/kg body weight, is used.
The present drug may be systemically administered by intravenous injection, intramuscular injection, intraperitoneal injection, oral administration, parenteral administration such as suppository or any other prior art means. As pharmaceutical preparations, there may be intended injectable or oral pharmaceutical preparations. Injectable pharmaceutical preparations may include, for example, injectable powders. In this instance, one or more of suitable water-soluble excipients such as mannitol, sucrose, maltose, glucose and fructose may be added and dissolved in water and the solution is poured portionwise into ampoules and freeze-dried and sealed to form pharmaceutical preparations. Oral pharmaceutical preparations may include conventional tablets, capsules, granules, fine granules, powders as well as enteric coated preparations.
In the treatment of bone fracture, it may be systemically administered or locally given by injection and others. For local administration, a carrier containing the present drug may be more preferably implanted in the area close to the fractured portion. In this case, natural polymeric substances such as collagen or fibrin glue, synthetic polymeric substances capable of being dispersed in living body such as poly lactated glycolic acid can be used for carriers. In plastic surgery, cosmetic surgery, bone transplantation or dental transplantation, the present agent may be applied by coating on the surface of the bone or tooth to be transplanted with an adhesive substance such as collagen paste or fibrin glue. And, it may be applied to the tissue, the bone or alveolar bone in the portion to which bone or tooth is to be transplanted. In transplantation of bone or tooth, artificial bone or artificial fang may be used which is composed of, for example, metals, ceramics, glass and other natural or artificial inorganic substances such as hydroxyapatite. In this case, it may be also feasible to form a core portion with a dense material and form a surface portion with a porous material such as hydroxyapatite and to penetre the present agent into the porous portion. Also, it is possible that the surface of artificial bone composed of a denser materials is roughened to keep the present agent over the said surface.